Highly inefficient transit of oligonucleotides from outside cells to the intracellular compartments where functional activity of oligonucleotides takes place is the most serious limitation to practical realization of a full potential of oligonucleotid-based therapies. Several classes of oligonucleotides therapeutics (ONT), including antisense oligonucleotides (ASO), hydrophobically modified siRNAs (hsiRNA), GalNAc conjugated siRNAs, and LNP formulated siRNAs have validated biological efficacy and are in clinic [1, 2]. In all cases, the fraction of oligonucleotides reaching the intended place of biological function is surprisingly low with the majority of molecules being trapped in wrong cellular compartments, resulting in low efficiency and clinically limiting toxicity. Concurrently, there is a significant nd rapidly expanding body of evidence demonstrating natural small RNA trafficking between cells as a means of intracellular communication. Given the extremely high efficacy of oligonucleotide-mediated gene silencing (25-100 molecules of Ago2 loaded siRNAs per cell [3]), exploiting the natural pathways of intracellular small RNA trafficking opens a possibility to dramatically improve the efficacy of ONTs. We hypothesize that exploiting natural, evolutionarily conserved mechanisms and pathways for trafficking of small RNAs across cellular boundaries is the way to fundamentally improve ONTs efficiency. The goal of this proposal is to elucidate the pathways and mechanisms involved in natural small RNA and ONTs trafficking and leverage this knowledge to increase the efficacy of oligonucleotide therapies. We have developed a unique set of experimental tools and analytical methods which enable us to systematically map and study endogenous small RNA as well as ONTs trafficking pathways. We have developed (1) a novel class of hydrophobic siRNAs (hsiRNAs), which efficiently enter cells and tissues; (2) an approach for visualization of native RNA trafficking through use of hsiRNA loaded exosomes as a model, (3) an approach for dissection of productive and non-productive ONTs trafficking using a panel of small molecules dramatically enhancing hsiRNAs efficacy by altering their cellular distribution; (4) a panel of four major classes of ONTs, which utilize different uptake mechanisms and silence a clinically relevant target huntingtin (htt), (5) a panel of engineered cell lines (GFP-fusions of trafficking markers) to study internalization pathways. Using these novel experimental tools along with TESM intracellular imaging, high resolution mass spectrometry and functional and biochemical analyses, we will identify the productive and non-productive cellular uptake mechanisms for both natural and synthetic oligonucleotides Completion of this project will result in (1) significant progress in our understanding of the natural mechanisms of extracellular uptake of small regulatory RNAs, (2) mapping uptake pathways for different classes of ONTs (3) development of the conceptual framework for generation of novel RNA chemistries relying on native uptake mechanisms. These findings will lead to a new generation of ONTs with dramatically improved clinical efficacy.